Jae Chul Cheong

Simultaneous determination of amphetamine-type stimulants and cannabinoids in fingernails by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the simultaneous detection and quantification of four amphetamine-type stimulants (amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA)) and two cannabinoids (Δ9-tetrahydrocannabinol (Δ9-THC) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH)) in fingernails. Fingernail clippings (30 mg) were washed with distilled water and methanol, and then incubated in 1.0 M sodium hydroxide at 95°C for 30 min. The compounds of interest were isolated by liquid-liquid extraction followed by derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) at 70°C for 15 min. The derivatized compounds were analyzed by GC-MS in the selective ion monitoring (SIM) mode. The linear ranges were 0.1–15.0 ng/mg for AP, 0.2–15.0 ng/mg for MDA, Δ9-THC and THCCOOH, and 0.2–30.0 ng/mg for MA and MDMA, with good correlation coefficients (r2 > 0.9991). The intra-day, inter-day, and inter-person precisions were within 10.6%, 6.3%, and 5.3%, respectively. The intra-day, inter-day and inter-person accuracies were between −6.1 and 5.0%, −6.2 and 5.7%, and −6.4 and 5.6%, respectively. The limits of detection (LODs) and quantification (LOQs) for each compound were lower than 0.056 and 0.2 ng/mg, respectively. The recoveries were in the range of 74.0–94.8%. Positive GC-MS results were obtained from specimens of nine suspected MA or cannabis abusers. The concentration ranges of MA, AP, and THCCOOH were 0.10–1.41, 0.12–2.64, and 0.20 ng/mg, respectively. Based on these results, the method proved to be effective for the simultaneous qualification and quantification of amphetamine-type stimulants and cannabinoids in fingernails.

Improved gas chromatography–negative ion chemical ionization tandem mass spectrometric method for determination of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair using mechanical pulverization and bead-assisted liquid–liquid extraction

A gas chromatography-negative ion chemical ionization tandem mass spectrometric (GC-NCI-MS/MS) method was developed and validated for the determination of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in human hair. After decontamination, hair samples were weighed (25mg), mechanically pulverized with a bead mill, and incubated in 0.7 mL of 1.0M sodium hydroxide at 95 °C for 30 min. Bead-assisted liquid-liquid extraction was performed with n-hexane:ethyl acetate (9:1, v/v), a method developed in our laboratory. The extract was evaporated to dryness, derivatized with pentafluoropropanol and pentafluoropropionic anhydride, and analyzed by GC-MS/MS in the negative ion chemical ionization mode using methane as the reagent gas. The linear ranges were 0.05-10.0 pg/mg for THC-COOH with the coefficient of determination (r(2) = 0.9976). The intra-day and inter-day precisions were within 1.7 and 13.8%, respectively. The intra-day and inter-day accuracies were -4.8 to 10.0% and -3.9 to 3.8%, respectively. The limit of detection and quantification were 0.015 and 0.05 pg/mg, respectively. The recoveries were in the range of 79.4-87.1%. The results indicate that the proposed method is simple, rapid, accurate, and precise for determination of THC-COOH in hair. The method identified THC-COOH in hair specimens from suspected marijuana abusers.

Rapid and sensitive determination of propofol glucuronide in hair by liquid chromatography and tandem mass spectrometry

A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantitation of propofol glucuronide in human hair has been developed and validated. Propofol glucuronide was extracted from 10mg of hair using a simple methanol extraction method, with recovery greater than 91% at 3 quality control samples (15, 100, 4000 pg/mg). A reversed phase column (C8) was used to analyze and the mobile phase was composed of ammonium formate and acetonitrile gradient at a flow rate of 0.2 mL/min. The lower limit of quantitation (LLOQ) was 5 pg/mg and the assay was linear to 5000 pg/mg. The intra- and inter-day precision (% CV, coefficient of variation) ranged from 1.26 to 4.50% while the accuracy (% RE, relative error) were -4.24 to 4.4%. The matrix effects were monitored at 3 different concentrations and the %CV of the results for these concentrations was less than 10.6%. Propofol glucuronide was stable during processing and analysis in human hair. The procedure was validated and applied to the analysis of hair samples in human subjects previously administered in propofol.

Rapid and Simple GC–MS Method for Determination of Psychotropic Phenylalkylamine Derivatives in Nails Using Micro-Pulverized Extraction*

Abstract:  A rapid and simple gas chromatography–mass spectrometry (GC–MS) method was developed and validated for the simultaneous detection and quantification of five psychotropic phenylalkylamines (amphetamine, methamphetamine, 3,4‐methylenedioxyamphetamine, 3,4‐methylenedioxymethamphetamine, and norketamine) in toenails. After external decontamination, nail clippings were mechanically pulverized with a bead mill and then incubated in methanol under ultrasonication at 50°C for 1 h. The resulting solutions were evaporated to dryness, derivatized, and analyzed by GC–MS. The intra‐ and inter‐day precisions were within 10.7% and 13.9%, respectively. The intra‐ and inter‐day accuracies were −4.2% to 5.0% and −2.4% to 8.4%, respectively. Limits of detection and quantification for each analyte were lower than 0.024 and 0.08 ng/mg, respectively. The recoveries were in the range of 80.6–87.5%. The results indicated that the proposed method is a simple, rapid, accurate, and precise for quantification of five phenylalkylamines in nails. The method was successfully applied to the simultaneous detection and quantification of phenylalkylamines in nail samples of possible drug abusers.

Gas chromatography‐mass spectrometric method for the screening and quantification of illicit drugs and their metabolites in human urine using solid‐phase extraction and trimethylsilyl derivatization

A simple and rapid GC-MS method has been developed for the screening and quantification of many illicit drugs and their metabolites in human urine by using automatic SPE and trimethylsilylation. Sixty illicit drugs, including parent drugs and their metabolites that are possibly abused in Korea, can be monitored by this method. Among them, 24 popularly abused illicit drugs were selected for quantification. Very delicate optimizations were carried out in SPE, trimethylsilylation derivatization, and GC/MS to enable such remarkable achievements. Trimethylsilylated analytes were well separated within 21 min by GC-MS. In the validation results, the LOD of all the analytes were in the range of 2-75 ng/mL. The LOQ of the quantified analytes were in the range of 5-98 ng/mL. The linearity (r(2)) of the quantified analytes ranged 0.990-1.000 in each concentration range between 10 and 1000 ng/mL. The mean recoveries ranged from 62 to 126% at three different concentrations of each analyte. The inter-day and inter-person accuracies were within -13.3 approximately 14.9%, and -10.1 approximately 13.0%, respectively, and the inter-day and inter-person precisions were less than 12.9%. The method was reliable and efficient for the screening and quantification of abused illicit drugs in routine urine analysis.

Simultaneous determination of methamphetamine, 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, N,N-dimethylamphetamine, and their metabolites in urine by liquid chromatography-electrospray ionization-tandem mass spectrometry

A liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the simultaneous detection and quantification of seven amphetamine derivatives (amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxy-N-amphetamine (MDA), 3,4-methylenedioxy-N-methamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), N,N-dimethylamphetamine (DMA), and N,N-dimethylamphetamine-N-oxide (DMANO)) in human urine. Seven deuterium-labeled compounds were prepared for use as internal standards to quantify the analytes. One milliliter of urine was combined with 1 mL of 0.2 M sodium carbonate buffer solution (pH 9.0) before solid phase extraction (SPE). An Oasis HLB SPE column followed by chromatographic separation on a Capcell Pak C18 MG-II column (150 × 2.0 mm I.D., 5 μm) and electrospray mass spectrometry with multiple reaction monitoring were used for selective and sensitive detection. The use of ammonium formate (5 mM, pH adjusted to 4.0 with formic acid, Solvent A) and acetonitrile (Solvent B) as the mobile phase at a flow rate of 230 μL/min was found to be the most effective for the separation. The linear ranges were 5.0–1000 ng/mL for AP, MDA, MDMA, MDEA, DMA, and DMANO and 10.0–1000 ng/mL for MA, with good correlation coefficients (r2 > 0.996). The intra-day, inter-day, and interperson precisions were within 14.6%, 12.1% and 15.5%, respectively. The intra-day, inter-day, and interperson accuracies were between −11.6 and 9.0%, −7.9 and 2.3%, and −13.2 and 4.3%, respectively. The limits of detection (LODs) for each analytical compound were lower than 1.95 ng/mL. The recovery ranged from 72.3 to 103.3%. The applicability of the developed method was examined by analyzing several urine samples from confirmed drug abusers.

AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 µM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 µM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

Reference ranges for urinary levels of testosterone and epitestosterone, which may reveal gonadal function, in a Korean male population

Cannabis, or marijuana, the most commonly used illicit drug in the world, has been shown to be responsible for suppressing the production and secretion of androgens, particularly testosterone. However, despite such findings in animals, the chronic effects of marijuana use on human endocrine systems have proved to be inconsistent. Here, we investigated the reference ranges of urinary levels of testosterone (T) and epitestosterone (E) as well as their metabolic ratio of T/E in a Korean male population (n=337), which would enable an evaluation of abnormal changes in steroid metabolism induced by habitually administered cannabis. The T/E ratio was significantly decreased in the marijuana group (n=18), while the urinary testosterone concentrations were also tended to decrease. This study is the first to provide data for the reference values of two urinary androgens and T/E values among control Korean males, and, furthermore, suggests that the T/E ratio, though not testosterone levels, might be used to understand the suppression of human male gonadal function affected by smoking marijuana.

Screening Method for the Detection of Methamphetamine in Hair Using Fluorescence Polarization Immunoassay

A hair screening method has been developed for the detection of methamphetamine using an immunoassay analyzer (AxSYM) with a fluorescence polarization immunoassay (FPIA) technique. The method consisted of washing, cutting and digesting a hair sample (5 mg) with an enzymatic digestion solution. The digested hair sample was centrifuged, and then an aliquot of the supernatant was used to conduct the screening. The results obtained from FPIA, in most cases, showed concentrations above 70.0 ng/mL of methamphetamine for hair samples that contained 0.5 ng/mg of methamphetamine, determined by gas chromatography-mass spectrometry (GC-MS). The percent sensitivity, defined as the true positive rate of screened and confirmed results, and the percent specificity, defined as the true negative rate of screened and confirmed results, of the FPIA screening method were 100.0 and 96.7% (false positive rate of 3.3%), respectively, when the threshold level for FPIA analysis was set at 70.0 ng/mL (n = 60).The correlation coefficient (r) for the linear relationship between FPIA and GC-MS results was 0.91 in real hair samples. The recommended amount of hair sample was found to be 5.0 mg for FPIA screening analysis when the concentration of methamphetamine in hair samples determined by GC-MS was found to be more than 0.5 ng/mg. The method developed in this study was reliable and effective for the screening of methamphetamine in routine hair analysis.